Department of Biotechnology, National Institute of Pharmaceutical Education and Research, Sector 67, S.A.S. Nagar, Punjab, 160062, India.
Protein Pept Lett. 2023;30(5):361-366. doi: 10.2174/0929866530666230307113055.
α-Synuclein, a natively disordered protein, is a key component of Lewy bodies, the ubiquitinated protein aggregates which are the pathological hallmark of Parkinson's disease (PD). Meclofenoxate (centrophenoxine) is a nootropic drug which has shown beneficial therapeutic effects in various neuronal diseases. Administration of meclofenoxate enhanced levels of dopamine and improved motor function in animal models of Parkinson's disease (PD). Evidence suggested that dopamine interacts with and modulates α-synuclein aggregation.
The aim of this work was to investigate whether the observed positive effect of addition of meclofenoxate, a nootropic agent, on dopamine level, could be correlated with its effect on aggregation of α-synuclein.
Purification of recombinant human α-synuclein was performed by anion exchange chromatography. The purified protein was incubated in the absence and presence of meclofenoxate and was analyzed for aggregation by Thioflavin T fluorescence spectroscopy. Conformational changes in α-synuclein were monitored by fluorescence spectroscopy and fluorescence quenching studies using a neutral quencher. Secondary structure analysis of α-synuclein was monitored by circular dichroism spectroscopy.
Recombinant human α-synuclein was expressed and purified by anion-exchange chromatography. Incubation of α-synuclein with meclofenoxate led to lowering aggregation in a concentration-dependent manner. Reduction in formation of oligomers was seen which suggested the formation of an off-pathway species which did not give rise to an aggregation-competent entity. Fluorescence quenching studies revealed that the additive distorted the native conformation of α- synuclein, leading to the formation of lower amounts of aggregation-prone species.
In the presence of higher concentrations of meclofenoxate, α-synuclein undergoes a change in its conformation. This change is not dependent on the concentration of the additive. This non-native conformer promotes the formation of a species which does not undergo further aggregation. Our study provides a mechanistic explanation of the earlier observation that meclofenoxate has a beneficial effect on progression of PD in animal models.
α-突触核蛋白是一种天然无序的蛋白质，是路易体的关键组成部分，路易体是帕金森病（PD）的病理标志，是泛素化蛋白聚集体。甲氯芬酯（centrophenoxine）是一种益智药，在各种神经疾病中显示出有益的治疗效果。在帕金森病（PD）的动物模型中，给予甲氯芬酯可提高多巴胺水平并改善运动功能。有证据表明，多巴胺与α-突触核蛋白的聚集相互作用并调节其聚集。
本工作旨在研究益智剂甲氯芬酯对多巴胺水平的观察到的积极影响是否与其对α-突触核蛋白聚集的影响相关。
通过阴离子交换色谱法对重组人α-突触核蛋白进行纯化。在不存在和存在甲氯芬酯的情况下孵育纯化的蛋白质，并通过硫黄素 T 荧光光谱法分析其聚集。通过荧光光谱和使用中性淬灭剂的荧光猝灭研究监测α-突触核蛋白的构象变化。通过圆二色性光谱监测α-突触核蛋白的二级结构分析。
通过阴离子交换色谱法表达和纯化了重组人α-突触核蛋白。α-突触核蛋白与甲氯芬酯孵育会导致聚集以浓度依赖性方式降低。观察到低聚物形成减少，这表明形成了一种非路径物种，不会产生具有聚集能力的实体。荧光猝灭研究表明，添加剂扭曲了α-突触核蛋白的天然构象，导致形成了较少的聚集倾向物种。
在较高浓度的甲氯芬酯存在下，α-突触核蛋白的构象发生变化。这种变化不依赖于添加剂的浓度。这种非天然构象促进了一种不会进一步聚集的物种的形成。我们的研究为先前观察到的甲氯芬酯在动物模型中对 PD 进展具有有益作用提供了机制解释。
